Tag: CRISPR

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CRISPR ‘Molecular Scissors’ can Leave Gaping Holes in the Chromosome

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CRISPR-Cas9 is a customisable tool that lets scientists cut and insert small pieces of DNA at precise areas along a DNA strand. This lets scientists study our genes in a specific, targeted way. Credit: Ernesto del Aguila III, National Human Genome Research Institute, NIH

The CRISPR molecular scissors have the potential to revolutionise the treatment of genetic diseases. This is because they can be used to correct specific defective sections of the genome. Unfortunately, there is a catch: under certain conditions, the repair can lead to new genetic defects – as in the case of chronic granulomatous disease. This was reported in the journal Communications Biology by a team from the University of Zurich (UZH).

Chronic granulomatous disease is a rare hereditary disease that affects about one in 120 000 people. The disease impairs the immune system, making patients susceptible to serious and even life-threatening infections. It is caused by the absence of two letters, called bases, in the DNA sequence of the NCF1 gene. This error results in the inability to produce an enzyme complex that plays an important role in the immune defence against bacteria and moulds.

The CRISPR tool works…

The research team has now succeeded in using the CRISPR system to insert the missing letters in the right place. They performed the experiments in cell cultures of immune cells that had the same genetic defect as people with chronic granulomatous disease. “This is a promising result for the use of CRISPR technology to correct the mutation underlying this disease,” says team leader Janine Reichenbach, professor of somatic gene therapy at the University Children’s Hospital Zurich and the Institute for Regenerative Medicine at UZH.

… but unfortunately, it’s not perfect

Interestingly however, some of the repaired cells now showed new defects. Entire sections of the chromosome where the repair had taken place were missing. The reason for this is the special genetic constellation of the NCF1 gene: it is present three times on the same chromosome, once as an active gene and twice in the form of pseudogenes. These have the same sequence as the defective NCF1 and are not normally used to form the enzyme complex.

CRISPR’s molecular scissors cannot distinguish between the different versions of the gene and therefore occasionally cut the DNA strand at multiple locations on the chromosome – at the active NCF1 gene as well as at the pseudogenes. When the sections are subsequently rejoined, entire gene segments may be misaligned or missing. The medical consequences are unpredictable and, in the worst case, contribute to the development of leukaemia. “This calls for caution when using CRISPR technology in a clinical setting,” says Reichenbach.

Safer method sought

To minimise the risk, the team tested a number of alternative approaches, including modified versions of CRISPR components. They also looked at using protective elements that reduce the likelihood of the genetic scissors cutting the chromosome at multiple sites simultaneously. Unfortunately, none of these measures were able to completely prevent the unwanted side effects.

“This study highlights both the promising and challenging aspects of CRISPR-based therapies,” says co-author Martin Jinek, a professor at the UZH Department of Biochemistry. He says the study provides valuable insights for the development of gene-editing therapies for chronic granulomatous disease and other inherited disorders. “However, further technological advances are needed to make the method safer and more effective in the future.”

Source: University of Zurich

Categories : GeneticsTags :

New, Modified CRISPR Protein can Fit inside Virus Used for Gene Therapy

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Novel enEbCas12a protein shows potential promise as gene-editing tool to one day treat disease

Researchers have developed a novel version of a key CRISPR gene-editing protein that shows efficient editing activity and is small enough to be packaged within a non-pathogenic virus that can deliver it to target cells. Hongjian Wang and colleagues at Wuhan University, China, present these findings May 30th in the open-access journal PLOS Biology.

Recent years have seen an explosion of research attempting to harness CRISPR gene-editing systems – which are found naturally in many bacteria as a defence against viruses – so they can be used as potential treatments for human disease. These systems rely on so-called CRISPR-associated (Cas) proteins, with Cas9 and Cas12a being the two most widely used types, each with their own quirks and strengths.

One promising idea is to package CRISPR proteins within a non-pathogenic virus, which could then deliver the proteins to target cells; there, they would modify specifically targeted DNA sequences to treat disease. However, the commonly used adeno-associated virus is small, and while some Cas9 proteins can fit inside, Cas12a proteins are typically too large.

Now, Wang and colleagues have identified a relatively small version of Cas12a, termed EbCas12a, that occurs naturally in a species of the Erysipelotrichia class of bacteria. By deliberately switching out one of the amino acid building blocks of the protein for another, they boosted its gene-editing efficiency. When applied to mammalian cells in a dish in the lab, this modified protein—dubbed enEbCas12a—shows gene-editing efficiency comparable to that of two other Cas12a proteins known for highly accurate gene editing.

The research team then demonstrated that enEbCas12a is small enough to be used for adeno-associated virus-based gene therapy. They modified enEbCas12a to target a specific cholesterol-associated gene, packaged it within the virus, and administered the virus to mice with high cholesterol. One month later, they found a significant reduction of blood cholesterol levels in the treated mice, compared to mice that did not receive the virus.

More research will be needed to determine if enEbCas12a could one day be used to address human disease. Nonetheless, these findings suggest it could be possible to use adeno-associated virus to deliver Cas12a proteins for gene therapy.

The authors add, “The novel compact enEbCas12a, along with its crRNA, can be packaged into an all-in-one AAV system for convenient gene editing in vitro and in vivo with high-fidelity, which can be very beneficial for future clinical applications and more tool developments including all-in-one AAV- based multi-gene editing, base editing, primer editing, etc.”

Provided by PLOS

Categories : Genetics OphthalmologyTags :

CRISPR Treatment Improves Vision in Inherited Retinal Degeneration

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Photo by Jeffrey Riley on Unsplash

About 79% of clinical trial participants experienced measurable improvement after receiving experimental, CRISPR-based gene editing that is designed to fix a rare form of blindness, according to a paper published in the New England Journal of Medicine.

“This trial shows CRISPR gene editing has exciting potential to treat inherited retinal degeneration,” said corresponding author Mark Pennesi, MD, PhD. “There is nothing more rewarding to a physician than hearing a patient describe how their vision has improved after a treatment. One of our trial participants has shared several examples, including being able to find their phone after misplacing it and knowing that their coffee machine is working by seeing its small lights.

Pennesi is an ophthalmologist and Oregon Health & Science University’s lead scientist for the Phase 1/2 BRILLIANCE trial, which evaluated the safety and effectiveness of EDIT-101, an experimental CRISPR-based gene editing treatment developed by Editas Medicine. The experimental treatment was designed to edit a mutation in the CEP290 gene, which provides instructions to create a protein that is critical for sight.

People with this gene mutation have a rare condition that is commonly called Leber Congenital Amaurosis, or LCA, Type 10, for which there is currently no Food and Drug Administration-approved treatment. LCA’s various types occur in about 2 or 3 out of 100 000 newborns.

The OHSU Casey Eye Institute treated the trial’s first participant in early 2020. That procedure also marked the first time that CRISPR had been used to edit genes within the human body, called in vivo gene editing.

The new paper describes the study’s findings through February 2023 and details how the trial’s 14 participants – 12 adults and two children – responded to receiving EDIT-101 in one eye. Key results include:

  • 11 participants, about 79%, showed improvement in at least one of four measured outcomes.
  • 6 participants, about 43%, showed improvement in two or more outcomes.
  • 6 participants, about 43%, reported improved vision-related quality of life.
  • 4 participants, about 29%, had clinically meaningful improvement in visual acuity, or how well they could identify objects or letters on a chart.
  • There were no serious adverse events related to the treatment.
  • Most adverse events were mild or moderate, and all have since been resolved.

Four specific outcomes were used to evaluate the experimental treatment’s effectiveness:

  • Visual acuity
  • How well participants did in a full-field test, which involves seeing coloured points of light while looking into a specialised device
  • How well participants navigated a research maze with physical objects and varying amounts of light
  • How much participants reported experiencing improved quality of life

Further research for a future treatment

In November 2022, trial sponsor Editas Medicine announced that it was pausing the trial’s enrolment and would seek another partner to continue the experimental therapy’s development. Pennesi and colleagues are exploring working with other commercial partners to conduct additional trials, in collaboration with Editas. The researchers hope future studies can examine ideal dosing, whether a treatment effect is more pronounced in certain age groups such as younger patients, and include refined endpoints to measure impacts on activities of daily living.

Source: Oregon Health & Science University

Categories : Cardiovascular Disease GeneticsTags :

A Single Gene-editing Infusion may Control Inherited High LDL Cholesterol

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CRISPR-Cas9 is a customisable tool that lets scientists cut and insert small pieces of DNA at precise areas along a DNA strand. This lets scientists study our genes in a specific, targeted way. Credit: Ernesto del Aguila III, National Human Genome Research Institute, NIH

A single infusion of a CRISPR-based gene-editing therapy significantly reduced low-density lipoprotein cholesterol (LDL-C, the ‘bad cholesterol’) in people who carry one gene for the inherited condition that results in very high LDL-C levels and a high risk of heart attack at an early age, according to findings presented at the American Heart Association’s Scientific Sessions 2023.

“Instead of daily pills or intermittent injections over decades to lower bad cholesterol, this study reveals the potential for a new treatment option – a single-course therapy that may lead to deep LDL-C lowering for decades,” said senior study author Andrew M. Bellinger, M.D., Ph.D., chief scientific officer at Verve Therapeutics in Boston.

The investigational treatment, VERVE-101, uses DNA-editing technology to permanently turn off the PCSK9 gene in the liver. PCSK9 is a gene that plays a critical role in controlling blood LDL-C through its regulation of the LDL receptor. People with heterozygous familial hypercholesterolaemia (ie, one gene for the disorder inherited from one parent) are treated with oral lipid-lowering medications such as statins as well as PCSK9 inhibitors to bring levels under control, though this only occurs in a small percentage of patients. The study presented is the first human trial of VERVE-101.

Earlier this year, the results of the researchers’ one-year animal study were published in Circulation. In that animal study, VERVE-101 lowered PSCK9 levels 67%-83% and LDL-C 49%-69%, depending on the dose. After a single dose, the reductions have now lasted 2.5 years, supporting the idea that VERVE-101 may potentially be an effective long-term or permanent treatment for high LDL-C.

The ongoing, first-in-human study included 7 men and 2 women in New Zealand or the United Kingdom: average age of 54 years; 8 white adults; and 1 Asian adult. Each participant was diagnosed with heterozygous familial hypercholesterolemia and had extremely high bad cholesterol levels (average measure of 201mg/dL) despite taking the maximum-tolerated LDL cholesterol-lowering medication.

“These numbers are consistent with the fact that, despite available treatments, only about 3% of patients living with heterozygous familial hypercholesterolemia globally have reached target treatment goals,” Bellinger said.

The majority of study participants had pre-existing severe coronary artery disease and had already experienced a heart attack, or undergone coronary bypass surgery or stenting to allow adequate blood flow to heart muscle. None were taking PCSK9 inhibitors while enrolled in the study.

Each participant received a single intravenous infusion of VERVE-101, with the first cohort (n=3) receiving a low dose of 0.1 mg/kg and other cohorts receiving escalating doses, after consultation with an independent safety monitoring board. The highest dose received was 0.6 mg/kg.

The study found that the highest-two VERVE-101 doses:

  • reduced LDL-C by 39% and 48% in the two participants receiving 0.45mg/kg of the drug and 55% in the sole participant receiving 0.6mg/kg;
  • reduced blood PCSK9 protein levels by 47%, 59% and 84% in the three participants receiving the 0.45 mg/kg or 0.6 mg/kg doses; and
  • reduced LDL-C at six months in the sole participant receiving 0.6mg/kg, with follow-up ongoing.

“We were thrilled to see that the previous testing we had done of VERVE-101 in animal models translated faithfully to these findings in humans,” Bellinger said.

Most adverse events encountered were mild and unrelated to treatment. Serious adverse cardiovascular events, specifically a cardiac arrest, a myocardial infarction and an arrhythmia, occurred in two patients who had underlying advanced coronary artery disease. “All safety events were reviewed with the independent data safety monitoring board, who recommended continuation of trial enrolment with no protocol changes required,” Bellinger said.

Studies involving a larger number of patients and with a control group will be required to fully document the efficacy and safety of VERVE-101, noted Bellinger.

The study is still enrolling patients to receive the highest-two doses of VERVE-101. After a year’s follow-up, each participant will go into a long-term follow-up study for an additional 14 years, as required by the FDA for all participants in any human genome editing trials.

Among the study’s limitations is that this is an interim report with a few participants who all received the treatment; therefore, no participants receiving an alternate treatment or no treatment were available for direct comparison. Results in the study were measured by reductions in LDL-C, not changes in the occurrence of heart attacks; however, LDL-C reduction is a well-known, validated endpoint among patients with heterozygous familial hypercholesterolaemia and coronary artery disease.

Source: American Heart Assoication

Categories : Genetics NeurologyTags :

CRISPR Untangles the Connections between Genome Organisation and Autism

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Photo by Peter Burdon on Unsplash

Using CRISPR gene editing, stem cells and human neurons, researchers have isolated the impact of a gene that is commonly mutated in autism. This new study, published today in The American Journal of Human Genetics, ties mutations in the gene CHD8 with a broad spectrum of molecular and cellular defects in human cortical neurons.

Autism is a highly heritable disorder with a recent increase in incidence – approximately 1 in 40 children in the US are diagnosed with autism. Over the past decade, sequencing studies have found many genes associated with autism but it has been challenging to understand how mutations in certain genes drive complex changes in brain activity and function.

The team, led by researchers at the New York Genome Center and New York University (NYU) and the Broad Institute, team developed an integrated approach to understand how mutations in the CHD8 gene alter genome regulation, gene expression, neuron function, and are tied to other key genes that play a role in autism. 

For more than a decade, it has been known that individuals with mutations in the CHD8 gene tend to have many similar ailments, such as autism, an abnormally large head size, digestive issues and difficulty sleeping. The CHD8 gene is a regulator of proteins called chromatin that surround the DNA but it is unclear how this particular gene might relate to major alterations in neural development and, in turn, result in autism. 

The research team identified numerous changes in physical state of DNA, which makes the genome more accessible to regulators of gene expression, and, in turn, drives aberrant expression of hundreds of genes. These molecular defects resulted in clear functional changes in neurons that carry the CHD8 mutation. These neurons are much less talkative: They are activated less often and send fewer messages across their synapses. 

The study authors initially observed these changes using human cortical neurons differentiated from stem cells where CRISPR was used to insert a CHD8 mutation. These findings were further bolstered by similar reductions in neuron and synapse activity when examining neurons from mice with a CHD8 mutation. These substantial defects in neuron function were circumvented when extra CHD8 was added to the cell using a gene therapy approach. In this case, extra copies of a healthy CHD8 gene without any mutation were added using a viral vector. Upon differentiation, the team found that the neurons rescued by the treatment returned to a normal rate of activity and synaptic communication, indicating that this gene therapy approach may be sufficient to restore function.

Lastly, when examining disrupted genes, the authors found that the CHD8 mutation seemed to specifically alter other genes that have been implicated in autism or intellectual disability, but not genes associated with unrelated disorders like cardiovascular disease. This suggest that CHD8 might influence selectively those genes that tend to be involved in neurodevelopmental disorders, providing an explanation for some of the particular characteristics of individuals carrying a CHD8 mutation.

Source: EurekAlert!

Categories : Diseases, Syndromes and Conditions GeneticsTags :

Scientists Snip Muscular Dystrophy Gene, Yielding Shorter but Now-functional Proteins

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CRISPR-Cas9 is a customisable tool that lets scientists cut and insert small pieces of DNA at precise areas along a DNA strand. This lets scientists study our genes in a specific, targeted way. Credit: Ernesto del Aguila III, National Human Genome Research Institute, NIH

The most common inherited muscular disorder and one of the most severe, Duchenne muscular dystrophy (DMD) results from mutations of the dystrophin gene. In the journal Stem Cell Reports, researchers used a dual CRISPR RNA method to restore dystrophin protein function in stem cells derived from DMD patients. By removing large sections of the dystrophin gene, the cells were able skip faulty or misaligned sections of the genetic code, yielding shortened but still functional proteins for a wide variety of mutation patterns associated with DMD.

“Dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein via multi-exon skipping induction,” says senior author Akitsu Hotta of Kyoto University. “We expect this study to enlighten new ways to treat DMD patients and other genetic disorders that require extensive deletions.”

Due to significant variations in the mutation patterns affecting the dystrophin gene, deleting a small section of the gene can only be used for a limited number of DMD patients. For example, the most common mono-exon skipping of exons 51, 53, and 45 can be applied to 13%, 8%, and 8% of DMD patients, respectively.

Multi-exon skipping (MES) has broad applicability to various DMD mutation patterns. By targeting the mutation hotspots in the dystrophin gene, MES from exon 45 to 55 was estimated to benefit more than 60% of DMD patients. Unfortunately, few techniques are available to induce a large deletion to cover the target exons spread over several hundred kilobases.

To overcome this hurdle, Hotta and his team used CRISPR-Cas3 to induce a deletion of up to 340 kilobases at the dystrophin exon 45-55 region in various DMD mutation patterns. Because it was rare to observe a deletion of more than a hundred kilobases using a single CRISPR RNA – which helps to locate the correct segment of DNA – the researchers used a pair of CRISPR RNAs inwardly sandwiching the target genomic region.

Limitations of the dual CRISPR RNA system include is variation in the deletion pattern, and the precise start and end points of the deletion cannot be fully controlled. This could be a drawback when a large but precise deletion is required. The study also did not demonstrate the functionality of the recovered dystrophin protein. Future research should aim to improve the overall genome editing efficiency of the Cas3 system.

“Our dual-Cas3 system might apply to future gene therapies once we’re able to deliver the dual-Cas3 components in vivo to skeletal muscle tissues safely and efficiently,” says Hotta. “The ability to induce several hundred kilobases of DNA deletion itself also has broad applicability for basic research when a large deletion is needed.”

Source: Science Daily

Categories : Genetics Medical Research & TechnologyTags :

New CRISPR Discovery Targets Infected Cells

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CRISPR-Cas9 is a customisable tool that lets scientists cut and insert small pieces of DNA at precise areas along a DNA strand. This lets scientists study our genes in a specific, targeted way. Credit: Ernesto del Aguila III, National Human Genome Research Institute, NIH

German and US scientists have discovered a CRISPR system in cells that shuts them down entirely to protect against viral replication, instead of merely chopping out foreign DNA that it comes across. It does this by shredding any DNA or RNA it comes across, causing the cell to become senescent and not become a virus factory. The newly identified CRISPR system is described in two papers published in Nature.

“With this new system, known as Cas12a2, we’re seeing a structure and function unlike anything that’s been observed in CRISPR systems to date,” says Jackson, assistant professor in Utah State’s Department of Chemistry and Biochemistry.

CRISPR, (Clustered Regularly Interspaced Short Palindromic Repeats) has taken science by storm with its gene-editing potential. Study of CRISPR DNA sequences and CRISPR-associated (Cas) proteins, which are actually bacterial immune systems, is still a young field.

Identified as a distinct immune system within the last five years, the Class 2, type V Cas12a2 is somewhat similar to the better-known ‘molecular scissors’ of CRISPR-Cas9, which binds to target DNA and cuts it, effectively shutting off a targeted gene. But CRISPR-Cas12a2 binds a different target than Cas9, and that binding has a very different effect.

Using cryo-electron microscopy, the team captured the CRISPR-Cas12a2 in a naturally occurring defensive strategy called abortive infection, a natural resistance strategy used by bacteria and archaea to limit the spread of viruses and other pathogens by preventing replication in the cell.

The team observed Cas12a2 in the act of cutting double-stranded DNA, bending it 90° to expose the backbone of the helix to cut it, a phenomenon that a phenomenon that elicits audible gasps from fellow scientists,” Jackson says.

Since the difference between a healthy cell and a malignant cell or infected cell is genetic, if Cas12a2 could be harnessed, “the potential therapeutic applications are significant.”

“If Cas12a2 could be harnessed to identify, target and destroy cells at the genetic level, the potential therapeutic applications are significant,” he says.

Source: Utah State University

Categories : Genetics HIVTags :

Curing HIV with a Dual Gene Editing Approach

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Source: Pixabay CC0

Gene editing therapy aimed at two targets – HIV-1 and CCR5, the co-receptor that helps the virus get into cells – can effectively eliminate HIV infection, report scientists in PNAS. This is the first to combine a dual gene-editing strategy with antiretroviral drugs to cure animals of HIV-1.

“The idea to bring together the excision of HIV-1 DNA with inactivation of CCR5 using gene-editing technology builds on observations from reported cures in human HIV patients,” said Kamel Khalili, PhD, professor at the Lewis Katz School of Medicine. “In the few instances of HIV cures in humans, the patients underwent bone marrow transplantation for leukaemia, and the donor cells that were used carried inactivating CCR5 mutations.”

Dr Khalili and Howard E. Gendelman, MD, professor at UNMC, were senior investigators on the new study from the Lewis Katz School of Medicine at Temple University and the University of Nebraska Medical Center (UNMC). The two researchers have been long-time collaborators and have strategically combined their research strengths to find a cure for HIV.

“We are true partners, and what we achieved here is really spectacular,” Dr Gendelman said. “Dr Khalili’s team generated the essential gene-editing constructs, and we then applied those constructs in our LASER-ART mouse model at Nebraska, figuring out when to administer gene-editing therapy and carrying out analyses to maximise HIV-1 excision, CCR5 inactivation, and suppression of viral growth.”

In previous work, Drs Khalili and Gendelman and their respective teams showed that HIV can be edited out from the genomes of live, humanised HIV-infected mice, leading to a cure in some animals. For that research, CRISPR gene-editing technology for targeting HIV-1 was combined with a therapeutic strategy known as long-acting slow-effective release (LASER) antiretroviral therapy (ART). LASER ART holds HIV replication at low levels for long periods of time, decreasing the frequency of ART administration.

Despite being able to eliminate HIV in LASER-ART mice, the researchers found that HIV could eventually re-emerge from tissue reservoirs and cause rebound infection. This effect is similar to rebound infection in human patients who have been taking ART but suddenly stop or experience a disruption in treatment. HIV integrates its DNA into the genome of host cells, it can lie dormant in tissue reservoirs for long periods of time, out of reach of antiretroviral drugs. As a consequence, when ART is stopped, HIV replication renews, giving rise to AIDS.

To prevent rebound infection, Dr Khalili and colleagues began work on next-generation CRISPR technology for HIV excision, developing a new, dual system aimed at permanently eliminating HIV from the animal model. “From success stories of human HIV patients who have undergone bone marrow transplantation for leukaemia and been cured of HIV, our hypothesis was that the loss of the virus’s receptor, CCR5, is important to permanently eliminating HIV infection,” he explained. They developed a simple and more practical procedure for the inactivation of CCR5 that includes an IV inoculation of the CRISPR gene editing molecule.

Experiments in humanized LASER-ART mice carried out by Dr Gendelman’s team showed that the constructs developed at Temple, when administered together, resulted in viral suppression, restoration of human T-cells, and elimination of replicating HIV-1 in 58% of infected animals. The findings support the idea that CCR5 has a key role in facilitating HIV infection.

The Temple team also anticipates soon testing the dual gene-editing strategy in non-human primates.

The new dual CRISPR gene-editing strategy holds exceptional promise for treating HIV in humans. “It is a simple and relatively inexpensive approach,” Dr Khalili noted. “The type of bone marrow transplant that has brought about cures in humans is reserved for patients who also have leukaemia. It requires multiple rounds of radiation and is not applicable in resource-limited regions, where HIV infection tends to be most common.”

Source: Temple University Health System

Categories : HIVTags :

A Step Closer to a Once-off Treatment for HIV

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HIV invading a human cell
HIV invading a human cell: Credit NIH

Researchers from Tel Aviv University have demonstrated success of a novel technology that may be developed into a one-time vaccine to treat people with HIV and AIDS. Using CRISPR technology, the researchers engineered B cells that in turn stimulate the immune system to produce HIV-neutralising antibodies.

Published in Nature, the study was led by Dr Adi Barzel and PhD student Alessio Nehmad and conducted in collaboration with additional researchers from Israel and the US.

“Based on this study,” said Dr Barzel, “we can expect that over the coming years we will be able to produce a medication for AIDS, additional infectious diseases and certain types of cancer caused by a virus, such as cervical cancer, head and neck cancer and more.”

He explains that the treatment can become a kind of permanent medication, lingering in the body to fight the virus. “We developed an innovative treatment that may defeat the virus with a one-time injection, with the potential of bringing about tremendous improvement in the patients’ condition. When the engineered B cells encounter the virus, the virus stimulates and encourages them to divide, so we are utilising the very cause of the disease to combat it. Furthermore, if the virus changes, the B cells will also change accordingly in order to combat it, so we have created the first medication ever that can evolve in the body and defeat viruses in the ‘arms race’.”

When they mature, the antibody-generating B cells move into the blood and lymphatic system and from there to the different body parts.

Dr Barzel explained: “Until now, only a few scientists, and we among them, had been able to engineer B cells outside of the body. In this study, we were the first to do this within body and then make those cells generate the desired antibodies. The genetic engineering is conducted with viral carriers derived from viruses that were also engineered. We did this to avoid causing any damage, and solely bring the gene coded for the antibody into the B cells in the body.”

“Additionally, in this case we have been able to accurately introduce the antibodies into a desired site in the B cell genome. All lab models that had been administered the treatment responded, and had high quantities of the desired antibody in their blood. We produced the antibody from the blood and made sure it was actually effective in neutralising the HIV virus in the lab dish.”

Source: Tel Aviv University

Categories : Cancer GeneticsTags :

CRISPR Editing can Destabilise the Genome, Study Finds

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DNA repair
Source: Pixabay/CC0

A new study published in Nature Biotechnology identifies risks in the use of CRISPR gene editing, which is employed in a number of therapies. Looking at its use in T cells, the researchers detected a loss of genetic material in a significant percentage – up to 10% of the treated cells. They explain that such loss can lead to destabilisation of the genome, which might cause cancer.

The study was led by Drs Adi Barzel, Dr Asaf Madi and Dr Uri Ben-David at Tel Aviv University.

Developed about a decade ago, CRISPR cleaves DNA sequences at certain locations in order to delete unwanted segments, or alternately repair or insert beneficial segments. It has already proved impressively effective in treating a range of diseases – cancer, liver diseases, genetic syndromes, and more. In 2020 at the University of Pennsylvania, the first approved clinical trial ever to use CRISPR took T cells from a donor, and expressed an engineered receptor targeting cancer cells, while using CRISPR to destroy genes coding for the original receptor – which otherwise might have caused the T cells to attack cells in the recipient’s body. 

In the present study, the researchers sought to examine whether the potential benefits of CRISPR therapeutics might be offset by risks resulting from the cleavage itself, assuming that broken DNA is not always able to recover.

Dr Ben-David and his research associate Eli Reuveni explained: “The genome in our cells often breaks due to natural causes, but usually it is able to repair itself, with no harm done. Still, sometimes a certain chromosome is unable to bounce back, and large sections, or even the entire chromosome, are lost. Such chromosomal disruptions can destabilise the genome, and we often see this in cancer cells. Thus, CRISPR therapeutics, in which DNA is cleaved intentionally as a means for treating cancer, might, in extreme scenarios, actually promote malignancies.”

To examine the extent of potential damage, the researchers repeated the 2020 Pennsylvania experiment, cleaving the T cells’ genome in exactly the same locations – chromosomes 2, 7, and 14. Using single-cell RNA sequencing, they analysed each cell separately and measured the expression levels of each chromosome in every cell.

They detected a significant loss of genetic material in some of the cells. For example, when chromosome 14 had been cleaved, about 5% of the cells showed little or no expression of this chromosome. When all chromosomes were cleaved simultaneously, the damage increased, with 9%, 10%, and 3% of the cells unable to repair the break in chromosomes 14, 7, and 2 respectively. The three chromosomes did differ, however, in the extent of the damage they sustained. 

Dr Madi and his student Ella Goldschmidt explained: “Single-cell RNA sequencing and computational analyses enabled us to obtain very precise results. We found that the cause for the difference in damage was the exact place of the cleaving on each of the three chromosomes. Altogether, our findings indicate that over 9% of the T-cells genetically edited with the CRISPR technique had lost a significant amount of genetic material. Such loss can lead to destabilisation of the genome, which might promote cancer.”

Based on their findings, the researchers caution that extra care should be taken when using CRISPR therapeutics. They also propose alternative, less risky, methods, for specific medical procedures, and recommend further research into two kinds of potential solutions: reducing the production of damaged cells or identifying damaged cells and removing them before the material is administered to the patient.

Dr Barzel and his PhD student Alessio Nahmad conclude: “Our intention in this study was to shed light on potential risks in the use of CRISPR therapeutics,” adding that as scientists, they “examine all aspects of an issue, both positive and negative, and look for answers.”

Source: EurekAlert!